Heavy Chain Constant Regions with Reduced Binding to Fc Gamma Receptors

ABSTRACT

The invention provides antibody heavy chain constant regions with a hinge region modified to reduce binding to Fcγ receptors. The modification occurs within positions 233-236 by replacement of natural residues by glycine(s) and/or deletion(s). Such modifications can reduce binding of an antibody bearing such a constant region to Fcγ receptors to background levels. The constant regions can be incorporated into any format of antibody or Fc fusion protein. Such antibodies or fusion proteins can be used in methods of treatment, particularly those in which the mechanisms of action of the antibody or Fc fusion protein is not primarily or at all dependent on effector functions, as is the case when an antibody inhibits a receptor-ligand interaction or agonizes a receptor.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/562,881, filed Sep. 28, 2017, which is a § 371 US national stage ofPCT/US2016/025051, filed Mar. 30, 2016, which claims the benefit under35 USC 119(e) of U.S. Provisional Application No. 62/140,350, filed Mar.30, 2015, each of which is incorporated by reference in its entirety forall purposes.

FIELD OF THE INVENTION

The present invention resides in the field of recombinant proteinengineering, and relates to optimized hinge variants of immunoglobulin(Ig) proteins, methods of engineering such Ig variants and suitabilityof such Ig variants in biopharmaceutical practice.

REFERENCE TO A SEQUENCE LISTING

This application incorporates by reference a sequence listing submittedin computer readable form as file 10140US02-Sequence.txt, created onDec. 19, 2019, and containing 78,858 bytes.

BACKGROUND

Antibodies of the IgG class are attractive therapeutic agents. IgGsexist as four subclasses in humans, IgG1, IgG2, IgG3, and IgG4. Theheavy chain constant (CH) region of IgG comprises three domains, CH1,CH2, CH3, and a hinge linking CH1 and CH2. Although the role of eachsubclass appears to vary between species, the heavy chain constantdomain is responsible for various biological effector functions. Thehuman IgG subclasses mediate several cellular immune responses throughtheir interaction with Fcγ (FcγRs), such as cell killing, phagocytosisand opsonization. Such interaction involves binding of at leastfunctional CH2 and CH3 domains of a heavy chain constant region to anFcγR on the surface of an effector cell, such as a natural killer cell,an activated macrophage or the like. Complement-mediated lysis can alsobe triggered by the interaction of the Fc region with various complementcomponents.

Effector functions are useful in some antibody therapies, such astreatment of some cancers or pathogens, in which effector function isprimarily or at least partially responsible for killing cancer cells orthe pathogen. However, other antibody therapies are mediated entirely orpredominantly by effector-independent mechanisms, such as inhibiting areceptor-ligand interaction or agonizing a receptor. In such therapies,antibody effector functions serve little or no useful purpose but canresult in undesired inflammation. In such circumstances, it may beadvantageous to engineer the Fc receptor binding properties of anantibody so as to inhibit some or all of the available effectormechanisms, without substantially affecting the antibody'spharmacokinetic properties, immunogenicity and variable regionsspecificity and affinity.

IgG heavy chain constant regions have been mutated in various positionsto test the effect of amino acids on IgG/FcγR interaction (see e.g.Canfield and Morrison, J Exp Med 73, 1483-1491 (1991); Chappel et al.JSC 268(33), 25124-31 (1993); and Armour et al., Eur. J. Immunol. 29,2613-24 (1999)). Several amino acid residues in the hinge region and inthe CH2 domain of a heavy chain constant region have been proposed asmediating binding to Fcγ receptors (see Sarmay et al., Mol Immunol 29,633-9 (1992); Greenwood et al., Eur. J. Immunol, 23(5), 1098 (1993),Morgan et al., Immunology 86, 319 (1995), Stevenson, ChemicalImmunology, 65, 57-72 (1997)). Glycosylation of a site (N297) in the CH2domain and variations in the composition of its carbohydrates alsostrongly affect the IgG/FcγR interaction (Stevenson, ChemicalImmunology, 65, 57-72 (1997); Siberil et al, Immunol. Ltrs. 106, 111-118(2006)).

Alanine residues have usually been the preferred substituent forreplacing a natural amino acid with an unnatural one so as to reducefunction because alanine has a side chain without any functional groups.For example, the well-known technique of alanine-scanning mutagenesissystematically replaces every natural residue in a protein or proteindomain with alanine to identify which natural residues contributeprimarily to function. Replacing an amino acid with a functional groupwith alanine eliminates the functional group and its contribution tobinding to any receptor, but the presence of the alanine side chainsubstantially preserves conformation, reducing the potential forimmunogenicity or other complexities due to conformational changes. Analternative strategy replaces amino acids in the hinge region of one IgGisotype with corresponding amino acids from human IgG2 isotype so as toreduce FcγR binding without unacceptable conformational changes andconsequent immunogenicity. The resulting chimeric Fc-containingantibodies include a substitution of EFLG at positions 232-236 with PVA(see WO14/121087).

SUMMARY OF THE CLAIMED INVENTION

The invention provides an immunoglobulin heavy chain comprising aconstant region, wherein positions 233-236 within a hinge domain are G,G, G and unoccupied; G, G, unoccupied, and unoccupied; G, unoccupied,unoccupied, and unoccupied; or all unoccupied, with positions numberedby EU numbering. Optionally, the immunoglobulin heavy chain of claim 1that is human IgG4 isotype. Optionally, positions 226-229 are CPPC.Optionally, the hinge domain amino acid sequence comprisesCPPCPAPGGG-GPSVF (SEQ ID NO:1), CPPCPAPGG--GPSVF (SEQ ID NO:2),CPPCPAPG---GPSVF (SEQ ID NO:3), or CPPCPAP----GPSVF (SEQ ID NO:4).Optionally, the constant region has an amino acid sequences comprisingSEQ ID NO:5, 6, 7 or 8 or a variant thereof having up to five insertionsdeletions, substitutions or insertions. Optionally, the constant regioncomprises SEQ ID NO: 5, 6, 7 or 8. Optionally, the constant regionconsists of SEQ ID NO: 5, 6, 7 or 8. Optionally, the immunoglobulinheavy chain comprises from N-terminal to C-terminal the hinge domain, aCH2 domain and a CH3 domain. Optionally, the immunoglobulin heavy chaincomprises from N-terminal to C-terminal a CH1 domain, the hinge domain,a CH2 domain and a CH3 domain. Optionally, the CH1 region, if present,remainder of the hinge region, if any, CH2 region and CH3 region are thesame human isotype. Optionally, the CH1 region, if present, remainder ofthe hinge region, if any, CH2 region and CH3 region are human IgG1.Optionally, the CH1 region, if present, remainder of the hinge region,if any, CH2 region and CH3 region are human IgG2. Optionally, the CH1region if present, remainder of the hinge region, if any, CH2 region andCH3 region are human IgG4. Optionally, the constant region has a CH3domain modified to reduce binding to protein A. Optionally, theimmunoglobulin heavy chain of any preceding claim linked at theN-terminus to a heavy chain variable region. Optionally, theimmunoglobulin heavy chain is duplexed with an immunoglobulin lightchain. Optionally, the immunoglobulin heavy chain is duplexed with animmunoglobulin light chain as a heterodimer comprising twoimmunoglobulin heavy chains and two light chains. The two heavy chainscan be the same or different. Optionally, the immunoglobulin heavy chainof any preceding claim linked at the N-terminus to a bindingpolypeptide. Optionally, the immunoglobulin heavy chain is linked via alinker to the binding polypeptide. Optionally, the binding polypeptideis an extracellular domain.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 shows correspondence of numbering schemes in the hinge region ofhuman IgG1, IgG2, and IgG4.

FIGS. 2-4 show the wild-type sequences of heavy chain constant regionsof isotypes IgG1, IgG2, IgG3 and IgG4 with delineation into CH1, hinge,CH2 and CH3 regions.

FIG. 5 is a schematic showing exemplary hinge-modified GGG-(233-236),GG--(233-236, G---(233-236) and no_G(233-236) replacement formatscompared with a previously described chimeric heavy chain constantregion, all of human IgG4 isotype.

FIGS. 6-10 show binding of various hinge-modified antibodies of humanIgG4 isotype (and wild-type IgG1 isotype antibody) to human FcγRI,FcγRIIA, FcγRIIB, FcγRIIIA, and FcRn.

FIG. 11A shows hinge-modified antibodies (as recovered from cell culturesupernatants) are unable to recruit human T-cells to lyse U937 cells,which bear FcγRI and FcγRIIA.

FIG. 11B also shows hinge-modified antibodies, that have been fullypurified, and that are unable to recruit human T-cells to lyse U937cells bearing FcγRI and FcγRIIA.

FIGS. 12A-D show that various hinge-modified antibodies show nosignificant activation of Jurkat cells with a luciferase marker when theactivation is dependent on anchoring of the antibody to HEK cellstransformed with FcγRIIA (FIG. 12A, FIG. 12B) or RIIB (FIG. 12C, FIG.12D). The activation by the positive control antibody in FIG. 12A andFIG. 12C is greatly reduced on competition with a blocking antibody, asin FIG. 12B and FIG. 12D.

FIG. 13 shows hinge-modified antibodies activating Jurkat cells with aluciferase marker when the antibodies are anchored to a plate surfaceinstead of by attempted binding to an FcγRIIA or RIIB receptor on theHEK cells.

FIG. 14 shows hinge-modified antibodies having variable regions thatbind CD20 display reduced antibody-dependent cellular cytotoxicity(ADCC) in the presence of NK cells cells engineered to express thehigher affinity V allele of FcγRIIIa and CD20-expressing Daudi cells.

DEFINITIONS

Antibodies or fusion proteins are typically provided in isolated form.This means that an antibody or fusion protein is typically at least 50%w/w pure of interfering proteins and other contaminants arising from itsproduction or purification but does not exclude the possibility that anantibody or fusion protein is combined with an excess of pharmaceuticalacceptable carrier(s) or other vehicle intended to facilitate its use.Sometimes antibodies or fusion proteins are at least 60, 70, 80, 90, 95or 99% w/w pure of interfering proteins and contaminants from productionor purification. Often an antibody or fusion protein is the predominantmacromolecular species remaining after its purification.

A basic antibody structural unit is a tetramer of subunits. Eachtetramer includes two identical pairs of polypeptide chains, each pairhaving one “light” (about 25 kDa) and one “heavy” chain (about 50-70kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. This variable region is initially expressed linkedto a cleavable signal peptide. The variable region without the signalpeptide is sometimes referred to as a mature variable region. Thus, forexample, a light chain mature variable region means a light chainvariable region without the light chain signal peptide. However,reference to a variable region does not mean that a signal sequence isnecessarily present; and in fact signal sequences are cleaved once theantibodies or fusion proteins of the invention have been expressed andsecreted. A pair of heavy and light chain variable regions defines abinding region of an antibody. The carboxy-terminal portion of the lightand heavy chains respectively defines light and heavy chain constantregions. The heavy chain constant region is primarily responsible foreffector function. In IgG antibodies, the heavy chain constant region isdivided into CH1, hinge, CH2, and CH3 regions. In IgA, the heavyconstant region is divided into CH1, CH2 and CH3. The CH1 region bindsto the light chain constant region by disulfide and noncovalent bonding.The hinge region provides flexibility between the binding and effectorregions of an antibody and also provides sites for intermoleculardisulfide bonding between the two heavy chain constant regions in atetramer subunit. The CH2 and CH3 regions are the primary site ofeffector functions and FcRn binding.

Light chains are classified as either kappa or lambda. Heavy chains areclassified as γ, mu, alpha, delta, or epsilon, and define the antibody'sisotype as IgG, IgM, IgA, IgD and IgE, respectively. Within light andheavy chains, the variable and constant regions are joined by a “J”segment of about 12 or more amino acids, with the heavy chain alsoincluding a “D” segment of about 10 or more amino acids. (See generally,Fundamental Immunology (Paul, W., ed., 2nd ed. Raven Press, N.Y., 1989),Ch. 7) (incorporated by reference in its entirety for all purposes).

The mature variable regions of each light/heavy chain pair form theantibody binding site. Thus, an intact antibody has two binding sites,i.e., is divalent. In natural antibodies, the binding sites are thesame. However, bispecific antibodies can be made in which the twobinding sites are different (see, e.g., Songsivilai and Lachmann, Clin.Exp. Immunol., 79:315-321 (1990); Kostelny et al., J. Immunol.,148:1547-53 (1992)). The variable regions all exhibit the same generalstructure of relatively conserved framework regions (FR) joined by threehypervariable regions, also called complementarity determining regionsor CDRs. The CDRs from the two chains of each pair are aligned by theframework regions, enabling binding to a specific epitope. FromN-terminal to C-terminal, both light and heavy chains comprise thedomains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of aminoacids to each domain is in accordance with the definitions of Kabat,Sequences of Proteins of Immunological Interest (National Institutes ofHealth, Bethesda, Md., 1987 and 1991), or Chothia & Lesk, J. Mol. Biol.196:901-917 (1987); Chothia et al., Nature 342:878-883 (1989). Kabatalso provides a widely used numbering convention (Kabat numbering) inwhich corresponding residues between different heavy chain variableregions or between different light chain variable regions are assignedthe same number. Although Kabat numbering can be used for antibodyconstant regions, the EU index is more commonly used, as is the case inthis application.

An antibody or fusion protein of the invention is mono-specific if allof its antigen (or ligand) binding regions have the same specificity. Anantibody or fusion protein is multispecific if its antigen bindingregions include at least two different specificities.

A hinge is a region of consecutive amino acid residues that connect theC-terminus of the C_(H)1 to the N-terminus of the C_(H)2 domain of animmunoglobulin. In human IgG1, IgG2 and IgG4, the hinge region runs fromresidue 216 to 236 by EU numbering. Residues 231-236 form a lower hingeand residues 216 to 230 form an upper and middle (or core) hinge. Thedemarcation between upper and middle varies by isotype. The upper andmiddle hinges of IgG1, IgG2 and IgG4 are 12-15 consecutive amino acidsencoded by a distinct hinge exon. The lower hinge includes severalN-terminal amino acids of the C_(H)2 domain (encoded by the C_(H)2 exon)(Brekkeet al. Immunology Today 16(2):85-90 (1995)). IgG3 comprises ahinge region consisting of four segments: one upper segment resemblingthe hinge region of IgG1, and 3 segments that are identical amino acidrepeats unique to IgG3.

The term “antibody” includes any form of antibody with at least onebinding region including monovalent fragments, divalent tetrameric unitsof two heavy chains and light chains, and higher order complexes of anyof these. An antibody can be mono-specific in which case all bindingregions have the same specificity or multi-specific in which the bindingsites have at least two specificities. Antibody fragments typicallyinclude a heavy chain variable region and a heavy chain constant regionand may also include a light chain variable region. For example, anantibody fragment can include from N-terminal to C-terminal a lightchain variable region, a peptide spacer, a heavy chain variable regionand a heavy chain constant region of the invention. Another fragmentincludes a heavy chain variable region (the binding region) and a heavychain constant region and no light chain (i.e., a Dab or nanobody).Likewise, a fusion protein includes a monomeric or dimeric fusionprotein unit, or higher order complexes.

A “monoclonal antibody” refers to a preparation of antibody moleculesresulting from propagation of a single clone consisting essentially ofthe same antibody molecules. Minor differences resulting fromspontaneous mutations arising in culture or posttranslational processingmay be present. A monoclonal antibody composition displays a singlebinding specificity and affinity for a particular epitope. Accordingly,the term “mouse or murine monoclonal antibody” refers to antibodiesdisplaying a single binding specificity which have variable and constantregions derived from murine or mouse germline immunoglobulin sequences.

A multispecific antibody typically comprises multiple different variabledomains (two in the case of bispecific antibody), wherein each variabledomain is capable of specifically binding to a separate antigen or to adifferent epitope on the same antigen. Exemplary bispecific formats thatcan be used with disclosed constant regions include e.g., scFv-basedbispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig,Quadroma, knobs-into-holes, common light chain (e.g., common light chainwith knobs-into-holes, etc.), CrossMab, CrossFab, (SEED)body, dualacting Fab (DAF)-IgG, and Mab2 bispecific formats (see, e.g., Klein etal. 2012, mAbs 4:6, 1-11, and references cited therein, for a review ofthe foregoing formats). Bispecific antibodies can also be constructedusing peptide/nucleic acid conjugation, e.g., wherein unnatural aminoacids with orthogonal chemical reactivity are used to generatesite-specific antibody-oligonucleotide conjugates which thenself-assemble into multimeric complexes with defined composition,valency and geometry. (See, e.g., Kazane et al. 2013, J. Am. Chem. Soc.9; 135(1):340-6 [Epub: Dec. 21, 2012]). Another exemplary multispecificformat that can be used with the disclosed constant regions includes afirst antigen-binding domain that specifically binds a target molecule,and a second antigen-binding domain that specifically binds aninternalizing effector protein, wherein such second antigen-bindingdomains are capable of activating and internalizing the effectorprotein, e.g. a receptor. (See US 2013/0243775A1.).

Binding refers to an interaction between at least two entities, ormolecular structures, such as an antibody-antigen interaction, or anFc-containing protein to an FcγR (wherein the Fc-containing protein isan antibody, Ig, antibody-binding fragment, or Fc-fusion protein, e.g.receptor-Fc fusion). For instance, binding affinity typicallycorresponds to a K_(D) value of about 10⁻⁷ M or less, such as about 10⁻⁸M or less, such as about 10⁻⁹ M or less when determined by, forinstance, surface plasmon resonance (SPR) technology in a BIAcore 3000instrument using the antigen or FcR as the ligand and the antibody, Ig,antibody-binding fragment, or Fc-containing protein as the analyte (orantiligand). Accordingly, an antibody or fusion protein binds to atarget antigen or receptor with an affinity corresponding to a K_(D)value that is at least ten-fold lower, such as at least 100 fold lower,for instance at least 1,000 fold lower, such as at least 10,000 foldlower, for instance at least 100,000 fold lower than its affinity forbinding to a non-specific antigen (e.g., BSA, casein). There is aninverse relationship between K_(D) and binding affinity, therefore thesmaller the K_(D) value, the higher the affinity. Thus, the term “loweraffinity” relates to a lower ability to form an interaction andtherefore a larger K_(D) value.

An epitope is an antigenic determinant capable of specific binding to anantibody. Epitopes usually consist of surface groupings of moleculessuch as amino acids or sugar side chains and usually have specific threedimensional structural characteristics, as well as specific chargecharacteristics. Conformational and nonconformational epitopes aredistinguished in that the binding to the former, but not the latter, islost in the presence of denaturing solvents. The epitope may compriseamino acid residues directly involved in the binding (also calledimmunodominant component of the epitope) and other amino acid residues,which are not directly involved in the binding, such as amino acidresidues which are effectively blocked by the specific antigen bindingpeptide (in other words, the amino acid residue is within the footprintof the specific antigen binding peptide).

A humanized antibody is a genetically engineered antibody in which theCDRs from a non-human “donor” antibody are grafted into human “acceptor”antibody sequences (see, e.g., Queen, U.S. Pat. Nos. 5,530,101 and5,585,089; Winter, U.S. Pat. No. 5,225,539, Carter, U.S. Pat. No.6,407,213, Adair, U.S. Pat. Nos. 5,859,205 6,881,557, Foote, U.S. Pat.No. 6,881,557). The acceptor antibody sequences can be, for example, amature human antibody sequence, a composite of such sequences, aconsensus sequence of human antibody sequences, or a germline regionsequence. Thus, a humanized antibody is an antibody having some or allCDRs entirely or substantially from a donor antibody and variable regionframework sequences and constant regions, if present, entirely orsubstantially from human antibody sequences. Similarly a humanized heavychain has at least one, two and usually all three CDRs entirely orsubstantially from a donor antibody heavy chain, and a heavy chainvariable region framework sequence and heavy chain constant region, ifpresent, substantially from human heavy chain variable region frameworkand constant region sequences. Similarly a humanized light chain has atleast one, two and usually all three CDRs entirely or substantially froma donor antibody light chain, and a light chain variable regionframework sequence and light chain constant region, if present,substantially from human light chain variable region framework andconstant region sequences. Other than nanobodies and dAbs, a humanizedantibody comprises a humanized heavy chain and a humanized light chain.A CDR in a humanized antibody is substantially from a corresponding CDRin a non-human antibody when at least 85%, 90%, 95% or 100% ofcorresponding residues (as defined by Kabat) are identical between therespective CDRs. The variable region framework sequences of an antibodychain or the constant region of an antibody chain are substantially froma human variable region framework sequence or human constant regionrespectively when at least 85, 90, 95 or 100% of corresponding residuesdefined by Kabat are identical.

Although humanized antibodies often incorporate all six CDRs (preferablyas defined by Kabat) from a mouse antibody, they can also be made withless than all CDRs (e.g., at least 3, 4, or 5 CDRs from a mouseantibody) (e.g., Pascalis et al., J. Immunol. 169:3076, 2002; Vajdos etal., Journal of Molecular Biology, 320: 415-428, 2002; Iwahashi et al.,Mol. Immunol. 36:1079-1091, 1999; Tamura et al, Journal of Immunology,164:1432-1441, 2000).

A chimeric antibody is an antibody in which the mature variable regionsof light and heavy chains of a non-human antibody (e.g., a mouse) arecombined with human light and heavy chain constant regions. Suchantibodies substantially or entirely retain the binding specificity ofthe mouse antibody, and are about two-thirds human sequence.

A veneered antibody is a type of humanized antibody that retains someand usually all of the CDRs and some of the non-human variable regionframework residues of a non-human antibody but replaces other variableregion framework residues that may contribute to B- or T-cell epitopes,for example exposed residues (Padlan, Mol. Immunol. 28:489, 1991) withresidues from the corresponding positions of a human antibody sequence.The result is an antibody in which the CDRs are entirely orsubstantially from a non-human antibody and the variable regionframeworks of the non-human antibody are made more human-like by thesubstitutions.

A human antibody refers to antibodies having variable and constantregions derived from human germline immunoglobulin sequences. Such anantibody can be one produced by a human or human B-cells, or atransgenic mouse bearing human immunoglobulin genes, or by from a phagedisplay, retroviral display, ribosomal display and the like (see forinstance Hoogenboom et al., J. Mol. Biol. 227, 381 (1991) (phagedisplay), Vaughan et al., Nature Biotech 14, 309 (1996) (phage display),Hanes and Plucthau, PNAS USA 94, 4937-4942 (1997) (ribosomal display),Parmley and Smith, Gene 73, 305-318 (1988) (phage display), Scott TIBS17, 241-245 (1992), Cwirla et al., PNAS USA 87, 6378-6382 (1990),Russell et al., Nucl. Acids Research 21, 1081-1085 (1993), Hogenboom etal., lmmunol. Reviews 130, 43-68 (1992), Chiswell and McCafferty,TIBTECH 10, 80-84 (1992), and U.S. Pat. No. 5,733,743). Human antibodiescan include amino acid residues not encoded by human germlineimmunoglobulin introduced by maturation in vivo, such as by somaticmutation or gene rearrangement in vivo. Human antibodies can alsoinclude a small number of mutations (e.g., up to 10 per heavy or lightchain) introduced by random or site-specific mutagenesis in vitro).

A transgenic animal for producing human antibodies refers to a non-humananimal having a genome comprising one or more human heavy and/or lightchain transgenes or transchromosomes (either integrated ornon-integrated into the animal's natural genomic DNA) and which iscapable of expressing fully human antibodies or at least antibodies withfully human variable regions. For example, a transgenic mouse can have ahuman light chain transgene and either a human heavy chain transgene orhuman heavy chain transchromosome, such that the mouse produces humanantibody when immunized with target antigen and/or cells expressing thetarget antigen. The human heavy chain transgene may be integrated intothe chromosomal DNA of the mouse, as is the case for transgenic mice,for instance HuMAb mice, such as HCo7 or HCol2 mice, or the human heavychain transgene may be maintained extrachromosomally, as is the case fortranschromosomal KM mice as described in WO02/43478. Such transgenic andtranschromosomal mice (collectively referred to as “transgenic mice”)are capable of producing multiple isotypes of human monoclonalantibodies to a given antigen (such as IgG, IgA, IgM, IgD and/or IgE) byundergoing V-D-J recombination and isotype switching. VELOCIMMUNE®genetically engineered mice comprise a replacement of unrearranged V(D)Jgene segments at endogenous mouse loci with human V(D)J gene segments.VELOCIMMUNE® mice express chimeric antibodies having human variabledomains and mouse constant domains (see, e.g., U.S. Pat. No. 7,605,237).Most other reports concern mice that express fully human antibodies fromfully human transgenes in mice that have disabled endogenousimmunoglobulin loci. The VELOCIMMUNE® mouse includes, in part, having agenome comprising human variable regions operably linked to endogenousmouse constant region loci such that the mouse produces antibodiescomprising a human heavy chain variable region and a mouse heavy chainconstant region in response to antigenic stimulation. The DNA encodingthe variable regions of the heavy chains of the antibodies can beisolated and operably linked to DNA encoding the human heavy chainconstant regions of the invention. The DNA can then be expressed in acell capable of expressing the fully human heavy chain of the antibody.

Several antibody effector functions are mediated at least in part by Fcreceptors (FcRs), which bind the Fc region of an antibody in theconstant domain (specifically, the CH2 and CH3 domain) of a typicalimmunoglobulin. There are a number of Fc receptors which are specificfor the different classes of immunoglobulins, i.e. IgG, IgE, IgA, IgM,and IgD. The human IgG Fc receptor family is divided into three groups:FcγRI (CD64), which is capable of binding IgG with high affinity, FcγRII(CD32) and FcγRIII (CD16) both of which are low affinity receptors. EachFcγR subclass is encoded by two or three genes, and alternative RNAsplicing leads to multiple transcripts, hence, a broad diversity in FcγRisoforms exists (e.g. FcγRIA (CD64; FCGR1A, Swiss Prot P12314), FcγRIB(CD64; FCRG1 B), FcγRIIA (CD32; FCGR2A, Swiss Prot P12318), FcγRIIB(CD32; FCGR2B, Swiss Prot P31994), FcγRIIC (CD32; FCGR2C), FcγRIIIA(CD16a; FCGR3A, Swiss Prot P08637), and FcγRIIIB (CD16b; FCGR3B)).Furthermore, Fc receptors are expressed on a variety of cells,including, e.g., B cells, monocytes, dendritic cells, neutrophils, andcertain lymphocytes. For example, U937 cells, a human monocyte cellline, express both FcγRI and FcγRIIA (see e.g., Jones, et al. J Immunol135(5):3348-53 (1985); and Brooks, et al. J Exp Med 170:1369-85 (October1989)).

Antibody-dependent cellular cytotoxicity or ADCC is an activity todamage a target cell when an Fcγ receptor-bearing cell (an immune cellor the like) binds to an Fc portion of a specific antibody through theFcγ receptor, when the specific antibody has attached to a cell-surfaceantigen of the target cell. Thus, ADCC is a mechanism by which Fcreceptor-positive effector cells can lyse target cells that haveadherent antigen-specific molecule. The ADCC activity can be evaluatedby for example measuring the fluorescent intensity using a fluorescentdye such as calcein AM (Wako Pure Chemical Industries, Ltd., 349-07201).When this approach is employed, the cytotoxic activity (% cell lysis)can be calculated, using the obtained values, according to the equation:(A−C)/(B−C)×100, wherein A is a fluorescent value in each sample, B isan average fluorescent value of the cells lysed and released into amedium with Nonidet P-40 having a final concentration of 1%, and C is anaverage fluorescent value when only the medium was added.

“Antibody-dependent cellular phagocytosis” or “ADCP” relates to effectorfunction that eliminates (or kills) a target cell by engulfing thetarget cell rather than inducing cytolysis. ADCP may be an important invivo mechanism for killing tumor cells. ADCP can be measured bytwo-color fluorescence flow cytometry methods, for example methodsutilizing, e.g. PKH2 (green fluorescent dye) andphycoerythrin-conjugated (red) monoclonal antibodies against differentcell surface proteins to differentiate the test cells, thus determiningphagocytic activity and rate of phagocytosis. Therapeutic strategiesthat selectively activate FcγRIIa relative to FcγRIIb may enhancemacrophage phagocytic activity (Richards et al. 2008 Mol. Cancer Ther.7(8):2517-27).

Complement-directed cytotoxicity or CDC refers to cytotoxic activity bythe complement system. CDC activity can be measured, for example thetarget cells, antibody, and complement solution (such as baby rabbitcomplement (Cedarlane Technologies)) are incubated and are allowed toreact, according to standard protocols (NIAID Manual of Tissue TypingTechniques 1979-1980, Edited by J. G. Ray, NIH Publication No.NIH-83-545.) The cytotoxic activity can be calculated in the same manneras the measurement of the ADCC activity. The cytotoxic activity can alsobe measured using a fluorescent dye (such as calcein) or radioactivedyes similarly to the above with respect to ADCC.

The term “subject” includes human and other mammalian subjects thatreceive either prophylactic or therapeutic treatment.

Compositions or methods “comprising” one or more recited elements mayinclude other elements not specifically recited. For example, acomposition that comprises antibody may contain the antibody alone or incombination with other ingredients.

DETAILED DESCRIPTION I. General

The invention provides antibody heavy chain constant regions with ahinge region modified to reduce binding to Fcγ receptors. Themodification occurs within positions 233-236 by EU numbering byreplacement of natural residues by glycine(s) and/or deletion(s). Theinventors unexpectedly found that such modifications in the hinge regionof antibodies can usefully reduce binding of such antibodies to Fcγreceptors to a greater extent than previous modifications in thisregion, and particularly can reduce binding to background levels for anyor all of FcγRI, FcγRIIA, FcγRIIB, and FcγRIIIA. These modifiedimmunoglobulin constant regions can be incorporated into virtually anyformat of antibody or Fc fusion protein. Such antibodies or fusionproteins can be used in methods of treatment, particularly those inwhich the mechanisms of action of the antibody or Fc fusion protein isnot primarily or at all dependent on effector functions, as is the casewhen an antibody inhibits a receptor-ligand interaction or agonizes areceptor.

II. Heavy Chain Constant Regions

The invention provides modified immunoglobulin heavy chain regions inwhich each of positions 233-236 by EU number is occupied by G or isunoccupied. Position 236 is unoccupied in canonical human IgG2 but isoccupied by in other canonical human IgG isotypes. Positions 233-235 areoccupied by residues other than G in all four human isotypes (see FIG.1). Position 233 is not believed to interact directly with Fcγ receptorsbut was included in the mutagenesis because removing replacing thewildtype glu residue in IgG1 and IgG4 or pro residue in IgG2 incombination with the other mutations would reduce immunogenicity. Infour exemplary modified constant regions, positions 233-236 are gly glygly unoccupied, gly gly unoccupied, unoccupied; gly, unoccupied,unoccupied, unoccupied and all unoccupied (see FIG. 5). These segmentscan be represented as GGG-, GG--, G--- or ---- with “-” representing anunoccupied position.

The hinge modification within positions 233-236 can be combined withposition 228 being occupied by P. Position 228 is naturally occupied byP in human IgG1 and IgG2 but is occupied by S in human IgG4 and R inhuman IgG3. An S228P mutation in an IgG4 antibody is advantageous instabilizing an IgG4 antibody and reducing exchange of heavy chain lightchain pairs between exogenous and endogenous antibodies.

Preferably positions 226-229 are occupied by C, P, P and C respectively.

Exemplary hinge regions have residues 226-236, sometimes referred to asmiddle (or core) and lower hinge, occupied by the modified hingesequences designated GGG-(233-236), GG--(233-236), G---(233-236) and noG(233-236).

hIgG1 CPPCPAPELLGGPSVF hIgG2 CPPCPAPPVA-GPSVF hIgG4 CPSCPAPEFLGGPSVFGGG-(233-236) CPPCPAPGGG-GPSVF (SEQ ID NO: 1) GG--(233-236)CPPCPAPGG--GPSVF (SEQ ID NO: 2) G---(233-236) CPPCPAPG---GPSVF(SEQ ID NO: 3) no_G(233-236) CPPCPAP----GPSVF (SEQ ID NO: 4)

The modified hinge regions described above can be incorporated into aheavy chain constant region, which typically include CH2 and CH3domains, and which may have an additional hinge segment (e.g., an upperhinge) flanking the designated region, and a CH1 region. Such additionalconstant region segments present are typically of the same isotype,preferably a human isotype, although can be hybrids of differentisotypes. The isotype of such additional human constant regions segmentsis preferably human IgG4 but can also be human IgG1, IgG2, or IgG3 orhybrids thereof in which domains are of different isotypes. Exemplarysequences of human IgG1, IgG2 and IgG4 are shown in FIGS. 2-4. Aconstant region is considered to be of a designated isotype if itdiffers from that isotype by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or10 substitutions, deletions or internal insertions, except however, thatthe CH1 domain can be omitted entirely as can the upper hinge region.CH1, CH2 and CH3 domains are considered to be of IgG1, IgG2 or IgG4isotype if differing from the CH1, CH2 and CH3 region of the exemplifiedsequence by no more than 1, 2, 3, 4 or 5 substitutions, deletions orinternal insertions. The remainder of a hinge outside the 226-236 regionsequences presented above is considered to be of IgG1, IgG2 or IgG4isotype if it differs from the corresponding part of the hinge region ofthe exemplified hinge sequences by no more than 1 or 2 substitutions,deletions or internal insertions.

Some preferred heavy chain constant regions have amino acid sequencesconsisting or comprising SEQ ID NO. 5, 6, 7 and 8. These heavy chainconstant regions incorporate the segments SEQ ID NO:1, SEQ ID NO:2, SEQID:3 and SEQ ID NO:4 at residues 226-236 shown above in anotherwise-human IgG4 isotype. Other preferred constant regions differfrom the designated SEQ ID NO. at up to 1, 2, 3, 4,5, 6, 7, 8, 9 or 10positions but retain at least GGG-, GG--, G--- or ----- at EU positions232-236 and P at position 228 and preferably retain of residues 226-236shown above for SEQ ID NO:1, SEQ ID NO:2, SEQ ID:3 and SEQ ID NO:4.Variations from the designated SEQ ID NOS. can represent one or severalnatural allotypic or isoallotypic variations, variations to increase orreduce an effector function such as complement-mediated cytotoxicity orADCC (see, e.g., Winter et al., U.S. Pat. No. 5,624,821; Tso et al.,U.S. Pat. No. 5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA103:4005, 2006), or to prolong half-life in humans (see, e.g., Hinton etal., J. Biol. Chem. 279:6213, 2004), for which exemplary substitutionsinclude a Gln at position 250 and/or a Leu at position 428 (EUnumbering. Other variations can add or remove sites ofpost-translational modification, such as N-linked glycosylation atN-X-S/T motifs. Variations can also include introduction of knobs (i.e.,replacement of one or more amino acids with larger amino acids) or holes(i.e., replacement of one or more amino acids with smaller amino acids)to promote formation of heterodimers between different heavy chains forproduction of bispecific antibodies. Exemplary substitutions to form aknob and hole pair are T336Y and Y407T respectively (Ridgeway et al.,Protein Engineering vol. 9 no. 7 pp. 617-621, 1996). Variations can alsoinclude mutations that reduce protein A interaction (e.g., H435R andY436F) in the EU numbering system. Bispecific antibodies in which oneheavy chain has such a variation, and another does not, can be separatedfrom their parental antibodies by protein-A affinity chromatography. Forexample, SEQ ID NOS. 9-12 are the same as SEQ ID NOS. 5-8 except for thepresence of H435R and Y436F mutations. One or more residues from theC-terminus of constant regions, particularly a C-terminal lysine on theheavy chain, can be lost as a result of post-translational modification.

Other heavy chain constant regions comprise or consist of SEQ ID NOS.16-19 and 20-23, which correspond to SEQ ID NOS. 5-8 and 9-12respectively except that the former are of human IgG1 rather than IgG4isotype. Other heavy chain constant regions comprise or consist of SEQID NOS. 24-27 and 28-31, which correspond to SEQ ID NOS. 5-8 and 9-12respectively except the former are of human IgG2 rather than IgG4isotype.

Other preferred constant regions differ from any of the above designatedSEQ ID NO. at up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 positions but retainat least GGG-, GG--, G--- or ----- at EU positions 232-236 and P atposition 228 and preferably retain residues 226-236 shown above for SEQID NO:1, SEQ ID NO:2, SEQ ID:3 and SEQ ID NO:4 in the same manner as wasdiscussed for IgG4 isotype constant regions.

Modified constant regions and antibodies or fusion proteinsincorporating such constant regions are characterized by reducedaffinity for Fcγ receptors compared with isotype matched controls(wildtype constant regions or antibodies or fusion proteinsincorporating the same). Binding affinity (Ka) is preferably reduced atleast 90, 95 or 99% compared with the isotype matched controls.Preferably binding affinity is reduced to background levels (i.e., samesignal within experimental error as in a control reaction with an sc-Fvfragment lacking any constant region or in which an irrelevant receptoris used in place of Fcγ. Preferably, affinity is reduced to a backgroundlevel or at least 90, 95 or 99% for each of human Fcγ receptors, γRI,γRIIA, γRIIB and γRIIIA.

Likewise, effector functions dependent on Fcγ receptor binding, such asADCC or ADCP are reduced, preferably by at least 90, 95 or 99% or morepreferably to background level. Such functions include cell killing orphagocytosis, B-cell activation, and release of inflammatory mediators,such as cytokines. Some such effects can be quantified by measurement ofEC50, which refers to the half maximal effective concentration of anantibody which induces a response halfway between the baseline andmaximum after a specified exposure time. The EC₅₀ essentially representsthe concentration of an antibody where 50% of its maximal effect isobserved. Some antibodies or fusion proteins including a modified heavychain constant region of the invention show cytotoxicity of less than20% cytolysis (i.e. % cytotoxicity), or less than 10%, or 5%, 4%, 3%,2%, or even 0% or undetectable cytolysis (cytotoxicity), as measured inan in vitro or ex vivo cell killing assay compared with suitableisotype-matched control antibodies with a wildtype constant region,optionally, measured at an antibody or fusion protein concentration of10 nM.

However, binding affinity of an antibody or fusion protein incorporatingsuch a heavy chain constant region is preferably not substantiallyaffected by the modified constant region. That is, the binding affinityis typically the same within experimental error or at least within afactor of 2 or 3 of a suitable control antibody with a isotype-matchedwild type constant region. The same is the case for functionalproperties not dependent on FcγR binding, such as ability to inhibitreceptor-ligand binding (e.g., EC50), or ability to agonize a receptor.

Immunogenicity of modified constant regions or antibodies or fusionproteins incorporated modified constant regions compared with isotypematched controls can be assessed in vitro from dendrocyte maturation orT-cell proliferation on challenge (Gaitonde et al., Methods Mol. Biol.2011; 716:267-80) or in vivo by comparing incidence of reactiveantibodies against administered antibodies between populations. Theimmunogenicity of modified constant regions or antibodies or fusionproteins incorporating the modified constant is preferably notsignificantly different from the isotype matched controls or not worsethan 2, 3, or 5 fold greater than the isotype matched control. Likewise,pharmacokinetic parameters such as Cmax, Cave rage, area under the curveand half-life are preferably not significantly different or at least notlower by a factor of no more than 2, 3 or 5 that isotype matchedcontrols. Such parameters can be measured in a mouse such as describedin the Examples, in other animal model or a human. Substantial retentionof such PK parameters provides an indication that modified constantregions or antibodies or fusion proteins incorporating them have notundergone substantial conformational changes triggering enhanced removalmechanisms.

III. Antibodies and Fusion Proteins

The modified heavy chain constant regions described above can beincorporated into antibodies or other fusion proteins. For example, forexpression of a monospecific antibody, a modified heavy chain constantregion is expressed fused to a heavy chain variable region and togetherwith a light chain including a light chain variable region and a lightchain constant region. The heavy and light chain bind to one another viathe CH1 region of the heavy chain and light chain constant region to aform a heterodimer. Two heterodimers then pair by association of hinge,CH2 and CH3 regions of the IgG heavy chain to form a tetramer unit, asis the case for a conventional antibody. For expression of a bispecificantibody, a modified heavy constant region is expressed fused to each oftwo heavy chain variable regions of different target specificities. Theheavy chains can each assembly with a co-expressed light chain and theheavy chain-light chain complexes form heterodimers in which both heavychains are present. The light chain variable regions can be the same(see e.g., US 20100331527A1) or different within a unit.

The modified constant regions can be used with any type of engineeredantibody including chimeric, humanized, veneered or human antibodies.The antibody can be a monoclonal antibody or a genetically engineeredpolyclonal antibody preparation (see U.S. Pat. No. 6,986,986).

For fusion protein proteins, a modified constant region is expressedlinked to a heterologous polypeptide. A heterologous polypeptide in afusion protein is a polypeptide not naturally linked to animmunoglobulin constant region. Such a polypeptide can be a full-lengthprotein or any fragment thereof of sufficient length to retain specificbinding to the antigen bound by the full-length protein. For example, aheterologous polypeptide can be a receptor extracellular domain orligand thereto. The heterologous polypeptide provides a binding regionat the N-terminus of the constant region and is sometimes referred tosimply as a binding region. The IgG CH1 region is not typically includedin the constant region for fusion proteins. The upper hinge region issometimes omitted or replaced by a synthetic linker peptide. Exemplaryreceptor proteins whose extracellular domains can be combined withmodified heavy chain constant regions of the invention are known in theart (see e.g. Klinkert, et al. J Neuroimmunol. 72(2): 163-8 (1997);Milligan et al., Curr Pharm. Des. 10(17): 1989-2001 (2004); and Schwache& Muller-Newen, Eur. J Cell Biol. 91 (6-7):428-34 (2012), doi:10.1016/j.ejcb.201 1.07.008. Epub 201 1 Sep. 29).

The binding region of a fusion protein can be any of the types ofbinding regions used in other fusion proteins produced to date (amongothers).

A multi-specific antibody or fusion protein can include bindingspecificities for an antigen on a target (e.g., a cancer cell orpathogen) and for an antigen on an effector cell (e.g., CD3 on aT-cell). Such a multi-specific complex forms a bridge between the targetcell and effector cell and promotes cytotoxic or opsonization activityof the effector cell. A multi-specific antibody or fusion protein canadditionally or alternatively include binding specificities for twodifferent antigens on the same target (e.g., a cancer cell or pathogen).Such an antibody or fusion protein can have greater selective toxicityto the target cell than an antibody or fusion protein with specificityfor a single antigen. Other multi-specific antibodies or fusion proteinsinclude binding regions for both a receptor and its ligand orcounter-receptor. Such antibodies or fusion proteins can exert greaterinhibition than antibodies or fusion proteins binding receptor orligand/counterreceptor alone. Any of these specificities and others canbe combined in the same multi-specific complex.

Antibodies or fusion proteins can also be chemically modified bycovalent conjugation to a polymer to, for instance, further increasetheir circulating half-life. Exemplary polymers, and methods to attachthem to peptides, are illustrated in for instance U.S. Pat. NoS.4,766,106, 4,179,337, 4,495,285 and 4,609,546. Additional illustrativepolymers include polyoxyethylated polyols and polyethylene glycol (PEG)(e.g., a PEG with a molecular weight of between about 1,000 and about40,000, such as between about 2,000 and about 20,000, e.g., about3,000-12,000 g/mol).

Antibodies or fusion proteins can be radiolabeled antibody for eitherdiagnostic or therapeutic purposes. Examples of radioisotopes include³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc, and ¹²⁵I, ¹³¹1, ¹⁸⁶Re, and ²²⁵Ac. Methodsfor preparing radiolabeled amino acids and antibodies or fusion proteinscontaining them are known (see for instance Junghans et al., in CancerChemotherapy and Biotherapy 655-686 (2nd edition, Chafner and Longo,eds., Lippincott Raven (1996)) and U.S. Pat. Nos. 4,681,581, 4,735,210,5,101,827, 5,102,990 (U.S. Pat. No. RE35,500), U.S. Pat. Nos. 5,648,471and 5,697,902. For example, a radioisotope may be conjugated by achloramine T method. Other detectable markers include an enzyme, achromophore, or a fluorescent label.

Antibodies or fusion proteins can be conjugated to a toxic agent. Toxicagents can be cytotoxic or cytostatic. Some example of toxic agentsinclude antitubulin agents, auristatins, DNA minor groove binders, DNAreplication inhibitors, alkylating agents (e.g., platinum complexes suchas cis-platin, mono(platinum), bis(platinum) and tri-nuclear platinumcomplexes and carboplatin), anthracyclines, antibiotics, antifolates,antimetabolites, chemotherapy sensitizers, duocarmycins, camptothecins,etoposides, fluorinated pyrimidines, ionophores, lexitropsins,nitrosoureas, platinols, pre-forming compounds, purine antimetabolites,puromycins, radiation sensitizers, steroids, taxanes, topoisomeraseinhibitors, vinca alkaloids, or the like.

IV. Antibody Expression

Nucleic acids encoding antibody chains or fusion proteins can be made bysolid state synthesis, PCR amplification of overlapping oligonucleotidefragments or site-directed mutagenesis of existing nucleic acids. Suchnucleic acids are expressed in an expression vector. Vectors can beconfigured to encode a modified heavy chain constant region and/or humanlight chain constant region such that they can be expressed as fusionswith inserted heavy chain and light chain variable regions or aheterologous polypeptide.

The origin of replication and expression control elements (promoter,enhancer, signal peptide and so forth) in a vector can be configured foruse in different cell types, such as bacteria, yeast or other fungi,insect cells, and mammalian cells. Mammalian cells are a preferred hostfor expressing nucleotide segments encoding antibodies or fusionproteins of the invention (see Winnacker, From Genes to Clones, (VCHPublishers, NY, 1987)). A number of suitable host cell lines capable ofsecreting intact heterologous proteins have been developed in the art,and include CHO cell lines, various COS cell lines, HeLa cells, HEK293cells, L cells, and non-antibody-producing myelomas including Sp2/0 andNS0. Preferably, the cells are nonhuman. Preferably, an antibody orfusion protein of the invention is expressed from a monoclonal cellline.

Expression vectors for these cells can include expression controlsequences, such as an origin of replication, a promoter, an enhancer(Queen et al., lmmunol. Rev. 89:49 (1986)), and necessary processinginformation sites, such as ribosome binding sites, RNA splice sites,polyadenylation sites, and transcriptional terminator sequences.Preferred expression control sequences are promoters derived fromendogenous genes, cytomegalovirus, SV40, adenovirus, bovinepapillomavirus, and the like. See Co et al., J. Immunol. 148:1149(1992).

Cells are transfected with one or more vectors encoding the antibody orfusion protein to be expressed. For a multi-chain antibody, the heavyand light chains can be expressed on the same or separate vectors. Forexpression of multi-specific complexes, the DNA encoding the componentsof the complexes (i.e., different antibodies or fusion proteins) can beon the same or different vectors.

Antibody or fusion protein chains are expressed, processed to removesignal peptides, assembled and secreted from host cells. Antibodies orfusion proteins can be purified from cell culture supernatants byconventional antibody purification methods. If the hybrid constantregion includes an IgG portion, then the purification can include achromatography step using protein A or protein G as the affinityreagent. Conventional antibody purification procedures, such as ionexchange, hydroxyapatite chromatograph or HPLC can also be used (seegenerally, Scopes, Protein Purification (Springer-Verlag, NY, 1982)).

V. Applications

Although antibodies and fusion proteins incorporating modified heavychain constant regions of the invention can be generally used in methodsof treatment or diagnosis, they are particularly useful in situations inwhich the mechanism of action of the antibody or fusion protein isentirely or at least predominantly independent of effector functions.Such is the case for example when the therapeutic objective is not tokill a target cell, but to inhibit or activate a cell surface moleculeon its surface without triggering cytotoxicity. Another setting in whichreduced binding to Fc receptors is desirable is when the antibody isbispecific, and its desired therapeutic properties arise from thedifferent binding specificities. For example, a common usage ofbispecific antibodies is to combine a tumor targeting specificity with aT cell activating specificity to trigger tumor-specific T cell killing.In this case, if the Fc portion binds to an Fc receptor, thenpotentially that could trigger undesirable killing of cells bearing Fcreceptors by T cells, or killing of T cells by Fc receptor-bearing cellssuch as natural killer cells or macrophages. Another setting in whichlack of effector functions can be advantageous is inhibiting aggregationof peptides that contribute to pathogenesis, such as in amyloidogenicdisease. A further setting is in vivo diagnosis, in which an antibody orfusion protein is intended to bind to a target but not result inclearing the target or cells bearing the target.

VI. Targets

Antibodies or fusion proteins incorporating a modified heavy chainconstant region may be directed to any number of cellular targetproteins. The antibodies or fusion proteins are particularly useful forsurface-bound target proteins. The desired response can be, for example,clearing of a target or cell or virus bearing the same, signaltransduction through a receptor, e.g., inducing apoptosis, inhibiting areceptor binding to a ligand or counterreceptor, or internalization ofan antibody or fusion protein conjugated to a toxic agent. Antibodies orfusion proteins can be made to the same targets as existing commercialantibodies or fusion proteins or can be derivatized versions ofcommercial antibodies or fusion proteins in which the existing constantregion has been replaced by a modified constant region of the presentinvention.

Targets of interest include growth factor receptors (e.g., FGFR, HGFR,PDGFR, EFGR, NGFR, and VEGFR) and their ligands. Other targets areG-protein receptors and include substance K receptor, the angiotensinreceptor, α and β adrenergic receptors, the serotonin receptors, and PAFreceptor. See, e.g., Gilman, Ann. Rev. Biochem. 56:625 649 (1987). Othertargets are CD (cluster of differentiation markers). Other targetsinclude ion channels (e.g., calcium, sodium, and potassium channels),muscarinic receptors, acetylcholine receptors, GABA receptors, glutamatereceptors, and dopamine receptors (see Harpold, U.S. Pat. Nos. 5,401,629and 5,436,128). Other targets are adhesion proteins such as integrins,selectins, and immunoglobulin superfamily members (see Springer, Nature346:425 433 (1990). Osborn, Cell 62:3 (1990); Hynes, Cell 69:11 (1992)).Other targets are cytokines, such as interleukins IL-1 through aboutIL-37 to-date, tumor necrosis factors, interferon, and, tumor growthfactor beta, colony stimulating factor (CSF) and granulocyte monocytecolony stimulating factor (GM-CSF). See Human Cytokines: Handbook forBasic &amp; Clinical Research (Aggrawal et al. eds., BlackwellScientific, Boston, Mass. 1991). Other targets are amyloidogenicpeptides, such as Abeta, alpha-synuclein or prion peptide. Other targetsare hormones, enzymes, and intracellular and intercellular messengers,such as, adenyl cyclase, guanyl cyclase, and phospholipase C. Targetmolecules can be human, mammalian or bacterial. Other targets areantigens, such as proteins, glycoproteins and carbohydrates frommicrobial pathogens, both viral and bacterial, and tumors.

Some examples of commercial antibodies and their targets includealemtuzumab (CD52); rituximab (CD20); trastuzumab (Her/neu);nimotuzumab, cetuximab (EGFR); bevacizumab (VEGF); palivizumab (RSV);abciximab (GpIIb/IIIa); infliximab, adalimumab, certolizumab, golimumab(TNF-alpha); baciliximab, daclizumab (IL-2); omalizumab (IgE);gemtuzumab (CD33); natalizumab (VLA-4); vedolizumab (alpha4beta7);belimumab (BAFF); otelixizumab, teplizumab (CD3); ofatumumab,ocrelizumab (CD20); epratuzumab (CD22); alemtuzumumab (CD52); eculizumab(C5); canakimumab (IL-1beta); mepolizumab (IL-5); reslizumab,tocilizumab (IL-6R); ustekinumab, briakinumab (IL-12, 23). Examples ofcommercial fusion proteins include etanercept which binds TNF-alpha,alefacept (LFA3-Fc fusion which binds CD2), TACI-Fc fusion which bindsBAFF and APRIL, abatacept (CTLA-4-Fc which binds CD80 and CD86), andromiplostim (a peptide analog of thrombopoietin fused to Fc). Any of thecommercial antibodies or fusion protein can be modified to replace theexisting heavy chain constant region with a modified heavy chainconstant region of the invention. Alternatively, a modified heavy chainconstant region can be linked to other antibodies with the same targetspecificity (e.g., as determined by a competition assay) as any of theabove commercial antibodies or fusion proteins.

VII. Methods of Treatment and Pharmaceutical Compositions

The antibodies and fusion proteins of the invention can also be used forsuppressing various undesirable immune responses including those for thesame therapies in which the commercial antibodies mentioned above havebeen used.

One category of immune disorders treatable by antibodies or fusionproteins of the invention is transplant rejection. When allogeneic cellsor organs (e.g., skin, kidney, liver, heart, lung, pancreas and bonemarrow) are transplanted into a host (i.e., the donor and donee aredifferent individual from the same species), the host immune system islikely to mount an immune response to foreign antigens in the transplant(host-versus-graft disease) leading to destruction of the transplantedtissue. The antibodies or fusion proteins are useful, inter alia, toblock alloantigen-induced immune responses in the donee.

A related use for antibodies or fusion proteins of the present inventionis in modulating the immune response involved in “graft versus host”disease (GVHD). GVHD is a potentially fatal disease that occurs whenimmunologically competent cells are transferred to an allogeneicrecipient. In this situation, the donor's immunocompetent cells mayattack tissues in the recipient. Tissues of the skin, gut epithelia andliver are frequent targets and may be destroyed during the course ofGVHD. The disease presents an especially severe problem when immunetissue is being transplanted, such as in bone marrow transplantation;but less severe GVHD has also been reported in other cases as well,including heart and liver transplants.

A further situation in which immune suppression is desirable is intreatment of autoimmune diseases such as type 1 diabetes, Crohn'sdisease, ulcerative colitis, multiple sclerosis, stiff man syndrome,rheumatoid arthritis, myasthenia gravis and lupus erythematosus. Inthese diseases, the body develops a cellular and/or humoral immuneresponse against one of its own antigens leading to destruction of thatantigen, and potentially crippling and/or fatal consequences. Autoimmunediseases are treated by administering one of the antibodies or fusionproteins of the invention. Other immune disorders treatable byantibodies or fusion proteins including modified constant regions of theinvention include asthma, allergies, celiac disease, psoriasis, anduveitis. Celiac disease, psoriasis and uveitis are autoimmune diseases.

The antibodies or fusion proteins of the invention can be used fortreating cancers in which a target antigen to which the antibody orfusion protein is expressed. The methods can be used to treat solidtumors, and particularly hematological malignancies, such as leukemia(e.g., T cell large granular lymphocyte leukemia), lymphoma (Hodgkin'sor Non-Hodgkin's), or multiple myeloma. Solid tumors include skin (e.g.,melanoma), ovarian, endometrial, bladder, breast, rectum, colon,gastric, pancreatic, lung, thymus, kidney and brain. Killing of cancercells can result from mechanism independent of FcγR bindings, such as byinduction of apoptosis, inhibition of a receptor-ligand interaction orby action of a conjugated cytotoxic moiety.

The antibodies or fusion protein can also be used for treatment ofpathogenic infections, such as viral, bacterial, protozoan or fungalinfection. Likewise killing can occur by mechanism independent of FcγRbinding such as by inhibiting an interaction between a pathogen and acell giving other elements of the immune system an opportunity to killthe pathogen or by action of a linked radionucleotide or toxin.

Antibodies or fusion proteins are administered in an effective regimemeaning a dosage, route of administration and frequency ofadministration that delays the onset, reduces the severity, inhibitsfurther deterioration, and/or ameliorates at least one sign or symptomof a disorder. If a subject is already suffering from a disorder, theregime can be referred to as a therapeutically effective regime. If thesubject is at elevated risk of the disorder relative to the generalpopulation but is not yet experiencing symptoms, the regime can bereferred to as a prophylactically effective regime. In some instances,therapeutic or prophylactic efficacy can be observed in an individualsubject relative to historical controls or past experience in the samesubject. In other instances, therapeutic or prophylactic efficacy can bedemonstrated in a preclinical or clinical trial in a population oftreated subjects relative to a control population of untreated subjects.

Exemplary dosages for an antibody or fusion protein are 0.01-20, or0.5-5, or 0.01-1, or 0.01-0.5 or 0.05-0.5 mg/kg body weight (e.g., 0.1,0.5, 1, 2, 3, 4 or 5 mg/kg) or 10-1500 mg as a fixed dosage. The dosagedepends on the condition of the subject and response to prior treatment,if any, whether the treatment is prophylactic or therapeutic and whetherthe disorder is acute or chronic, among other factors.

Administration can be parenteral, intravenous, oral, subcutaneous,intra-arterial, intracranial, intrathecal, intraperitoneal, topical,intranasal or intramuscular. Administration into the systemiccirculation by intravenous or subcutaneous administration is preferred.Intravenous administration can be, for example, by infusion over aperiod such as 30-90 min.

The frequency of administration depends on the half-life of the antibodyor fusion protein in the circulation, the condition of the subject andthe route of administration among other factors. The frequency can bedaily, weekly, monthly, quarterly, or at irregular intervals in responseto changes in the subject's condition or progression of the disorderbeing treated. An exemplary frequency for intravenous administration isbetween weekly and quarterly over a continuous cause of treatment,although more or less frequent dosing is also possible. For subcutaneousadministration, an exemplary dosing frequency is daily to monthly,although more or less frequent dosing is also possible.

The number of dosages administered depends on whether the disorder isacute or chronic and the response of the disorder to the treatment. Foracute disorders or acute exacerbations of chronic disorders between 1and 10 doses are often sufficient. Sometimes a single bolus dose,optionally in divided form, is sufficient for an acute disorder or acuteexacerbation of a chronic disorder. Treatment can be repeated forrecurrence of an acute disorder or acute exacerbation. For chronicdisorders, an antibody can be administered at regular intervals, e.g.,weekly, fortnightly, monthly, quarterly, every six months for at least1, 5 or 10 years, or the life of the subject.

Pharmaceutical compositions for parenteral administration are preferablysterile and substantially isotonic and manufactured under GMPconditions. Pharmaceutical compositions can be provided in unit dosageform (i.e., the dosage for a single administration). Pharmaceuticalcompositions can be formulated using one or more physiologicallyacceptable carriers, diluents, excipients or auxiliaries. Theformulation depends on the route of administration chosen. Forinjection, antibodies can be formulated in aqueous solutions, preferablyin physiologically compatible buffers such as Hank's solution, Ringer'ssolution, or physiological saline or acetate buffer (to reducediscomfort at the site of injection). The solution can containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively antibodies can be in lyophilized form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

Treatment with antibodies of the invention can be combined with othertreatments effective against the disorder being treated. For treatmentof immune disorders, conventional treatments include mast celldegranulation inhibitors, corticosteroids, nonsteroidalanti-inflammatory drugs, and stronger anti-inflammatory drugs such asazathioprine, cyclophosphamide, leukeran, FK506 and cyclosporine.Biologic anti-inflammatory agents, such as Tysabri® (natalizumab) orHumira® (adalimumab), can also be used. When used in treating cancer,the antibodies of the invention can be combined with chemotherapy,radiation, stem cell treatment, surgery or treatment with otherbiologics such as Herceptin® (trastuzumab) against the HER2 antigen,Avastin® (bevacizumab) against VEGF, or antibodies to the EGF receptor,such as (Erbitux®, cetuximab), and Vectibix® (panitumumab). Chemotherapyagents include chlorambucil, cyclophosphamide or melphalan,carboplatinum, daunorubicin, doxorubicin, idarubicin, and mitoxantrone,methotrexate, fludarabine, and cytarabine, etoposide or topotecan,vincristine and vinblastine. For infections, treatment can be incombination with antibiotics, anti-virals, anti-fungal or anti-protozoanagents or the like.

VIII. Other Applications

The antibodies or fusion proteins can be used for detecting their targetmolecule in the context of clinical diagnosis or treatment or inresearch. For example, the antibodies can be used to detect acancer-related antigen as an indication a subject is suffering from animmune mediated disorder amenable to treatment. The antibodies can alsobe sold as research reagents for laboratory research in detectingtargets and their response to various stimuli. In such uses, antibodiesor fusion proteins can be labeled with fluorescent molecules,spin-labeled molecules, enzymes or radioisotypes, and can be provided inthe form of kit with all the necessary reagents to perform the assay.The antibodies or fusion protein can also be used to purify their targetantigens e.g., by affinity chromatography.

Presence of labeled antibodies or fusion may be detected in vivo fordiagnosis purposes. In one embodiment, diagnosis comprises: a)administering to a subject an effective amount of a labeled antibody orfusion protein; b) waiting for a time interval following administrationfor permitting labeled antibody or fusion protein to concentrate atsites where antigen may be detected and to allow for unbound labeledantibody to be cleared to background level; c) determining a backgroundlevel; and d) detecting the labeled antibody or fusion protein in thesubject, such that detection of labeled antibody above the backgroundlevel is indicative that the subject has the disease or disorder, or isindicative of the severity of the disease or disorder. In accordancewith such embodiment, the antibody or fusion protein is labeled with animaging moiety suitable for detection using a particular imaging systemknown to those skilled in the art. Background levels may be determinedby various methods known in the art, including comparing the amount oflabeled antibody detected to a standard value previously determined fora particular imaging system. Methods and systems that may be used in thediagnostic methods of the invention include, but are not limited to,computed tomography (CT), whole body scan such as positron emissiontomography (PET), magnetic resonance imaging (MRI), and sonography.

All patent filings, websites, other publications, accession numbers andthe like cited above or below are incorporated by reference in theirentirety for all purposes to the same extent as if each individual itemwere specifically and individually indicated to be so incorporated byreference. If different versions of a sequence are associated with anaccession number at different times, the version associated with theaccession number at the effective filing date of this application ismeant. The effective filing date means the earlier of the actual filingdate or filing date of a priority application referring to the accessionnumber if applicable. Likewise if different versions of a publication,website or the like are published at different times, the version mostrecently published at the effective filing date of the application ismeant unless otherwise indicated. Any feature, step, element,embodiment, or aspect of the invention can be used in combination withany other unless specifically indicated otherwise. Although the presentinvention has been described in some detail by way of illustration andexample for purposes of clarity and understanding, it will be apparentthat certain changes and modifications may be practiced within the scopeof the appended claims.

EXAMPLES Example 1 Expression of Hinge-Modified Antibodies

Several of the antibodies described in the following examples arebispecific antibodies in which one arm is that of an anti-hCD3 antibodyand the other arm is from an anti-hCD20 antibody as described byWO2014047231.

Antibody 1, 9F7_VH_IgG4_GGG-(233-236), was made by site-directedmutagenesis using QuikChange Lightning Site-Directed Mutagenesis Kit(Agilent Technologies, Inc.; Catalog#210518) following themanufacturer's protocol, and starting with a chimeric IgG4 CH region(which contained the modifications S228P and ELLG233-236PVA; seeWO2014047231). After sequence confirmation, the coding region was movedto a parent vector using Xho1-Not1 restriction sites to avoid anymutations in the non-coding region, and subsequently generating a vectorconstruct with the hinge-modified IgG4 CH, having the hingemodifications of SEQ ID NO:1.

The anti-CD3(9F7 VH; see “L2K” based on WO2004/106380) variable regionnucleic acid sequence was amplified and introduced into the same plasmidas the hinge-modified IgG4 S228P, GGG-(233-236) and the sequence wasconfirmed using PCR. The final plasmid was used to produce Antibody 1using standard cell culture methodologies for isolating antibodies.

Antibody 2, 9F7_VH_IgG4_GG-(233-236), was made analogously to Antibody 1by using site directed mutagenesis to generate a vector construct withthe hinge modified IgG4 CH with GG--(233-236) (SEQ ID NO:2). Theanti-CD3(9F7 VH; see WO2004/106380) variable region nucleic acidsequence was amplified and introduced into the same plasmid usingstandard molecular biology methods. Ab 2 was isolated using standardmethodologies.

Antibody 3, 9F7_VH_IgG4_G-(233-236), was made analogously to Antibody 1by using site directed mutagenesis to generate a vector construct withthe hinge modified IgG4 CH with G---(233-236) (SEQ ID NO:3). Theanti-CD3 (9F7 VH; see WO2004/106380) variable region nucleic acidsequence was amplified and introduced into the same plasmid usingstandard molecular biology methods. Ab 3 was isolated using standardmethodologies.

Antibody 4, 9F7_VH_IgG4_No_G(233-236), was made analogously to Antibody1 by using site directed mutagenesis to generate a vector construct withthe hinge modified IgG4 CH with No_G(233-236) (SEQ ID NO:4). Theanti-CD3(9F7 VH; see WO2004/106380) variable region nucleic acidsequence was amplified and introduced into the same plasmid. Ab 4 wasisolated using standard methodologies.

Antibody 5, 3B9_VH_IgG4_GGG-(233-236), was made by site-directedmutagenesis using QuikChange Lightning Site-Directed Mutagenesis Kit(Agilent Technologies, Inc.; Catalog#210518) following themanufacturer's protocol, and starting with a chimeric IgG4 CH region(which contained the modifications S228P and ELLG233-236PVA; seeWO2014047231). Antibody 5 is a monospecific, bivalent anti-CD20antibody, and the anti-CD20 variable region nucleic acid sequence (3B9VH) was isolated using standard methodologies as described inWO2014047231.

Antibody 6, 3B9_VH_IgG4_GG--(233-236), was made analogously to Antibody5 by using site directed mutagenesis to generate a vector construct withthe hinge modified IgG4 CH with GG--(233-236) (SEQ ID NO:2). Theanti-CD20 (3B9 VH; see WO2014047231) variable region nucleic acidsequence was isolated using standard methodologies.

Antibody 7, 3B9_VH_IgG4_G---(233-236), was made analogously to Antibody5 by using site directed mutagenesis to generate a vector construct withthe hinge modified IgG4 CH with G---(233-236) (SEQ ID NO:3). Theanti-CD20 (3B9 VH; see WO2014047231) variable region nucleic acidsequence was isolated using standard methodologies.

Antibody 8, 3B9_VH_IgG4_No_G(233-236), was made analogously to Antibody5 by using site directed mutagenesis to generate a vector construct withthe hinge modified IgG4 CH with No_G(233-236) (SEQ ID NO:4). Theanti-CD20 (3B9 VH; see WO2014047231) variable region nucleic acidsequence was isolated using standard methodologies.

Antibody 9, anti-transmembrane (TM) protein variable domains (B6H12.2,obtained from BioXCell, Cat. No. BE0019-1) were cloned in to a plasmidcontaining the hinge-modified IgG4 S228P, GGG-(233-236) nucleic acid, bymethods similarly described for Antibody 1.

Antibody 10, B6H12.2_VH_IgG4_PVA was made according to protocolsdescribed herein, having a chimeric IgG4(S228P and ELLG233-236PVA) Fcdomain.

Antibody 11, anti-FELD1_VH_IgG4_PVA antibody, is isotype matched toAntibody 10, having a chimeric IgG4(S228P and ELLG233-236PVA) Fc domain.

Control Antibody A (Lot-L2), 9F7_VH_IgG4, is an antiCD3 (9F7 VH; seeWO2004/106380) human IgG4 isotype antibody (except having a CH3*mutation 435R and 436F, designated the star mutation—seeUS20100331527A1).

Control Antibody B (Lot-L2), 9F7_VH_IgG1, is an antiCD3 (9F7 VH; seeWO2004/106380) human IgG1 isotype antibody.

Control Antibody C: 9F7_VLx3B9_VH_IgG4, was made according to theprotocols described in WO2014047231. Control Ab C is a bispecificantiCD3xanti-CD20 monoclonal antibody having a wild-type IgG4 heavychain (except one arm has the star mutation in the CH3 region for easeof bispecific antibody isolation).

Control Antibody D (Lot-L5): 9F7_VLx3B9_VH_IgG4_PVA, was made accordingto the protocols described in WO2014047231. Control Ab D is a bispecificanti-CD3xanti-CD20 monoclonal antibody having a modified IgG4 heavychain [chimeric IgG4(S228P and ELLG233-236PVA), except one heavy chainhas the star mutation in the CH3 region for ease of bispecific antibodyisolation].

Control Antibody E: Anti-FeID1 monoclonal antibody binds a felineantigen with no cross-reactivity to human CD20 or CD3. This IgG1non-specific antibody control was obtained by methods described in PCTPublication No. WO2013/166236, published on Nov. 7, 2013.

Control Antibody F: is small batch (supernatant) preparation of 9F7(anti-CD3) with the chimeric IgG4 Fc (S228P and ELLG233-236PVA) (Seealso WO2014047231).

Control Ab G: 9F7_VH_IgG4_PVA was made according to the protocolsdescribed in WO2014047231 (Lot#2, purified). Control Ab G is amonospecific anti-CD3 monoclonal antibody having a modified IgG4 heavychain [chimeric IgG4(S228P and ELLG233-236PVA), except one heavy chainhas the star mutation in the CH3 region for ease of bispecific antibodyisolation].

Control Ab H (Lot #02-091210): 3139_VH_IgG1 is an anti-CD20 (3B9 VH; seeWO2004/106380) human IgG1 isotype antibody.

Control Ab I (Lot #01-110607): 3139_VH_IgG4 is an anti-CD20 (3B9 VH; seeWO2004/106380) human IgG4 isotype antibody.

Control Ab J (Lot #L1): 9F7_VK x3B9_VH_IgG4_PVA was made according tothe protocols described in WO2014047231. Control Ab J is a bispecificanti-CD3xanti-CD20 monoclonal antibody having a modified IgG4 heavychain [chimeric IgG4 (S228P and ELLG233-236PVA), except one heavy chainhas the star mutation in the CH3 region for ease of bispecific antibodyisolation].

Example 2 Loss of Affinity to Fcγ Receptors

The hinge-modified antibodies (i.e. GGG-, GG--, G--- or no-G hingereplacement; Antibodies 1-4) and various control antibodies were testedfor binding affinity to Fcγ receptors by surface plasmon resonance(SPR). The controls included Control Ab D (antiCD3xantiCD20-sIgG4 (S228Pand ELFG233-236PVA), Control Ab B (antiCD3, human IgG1 isotype), andControl Ab A (antiCD3, human IgG4 isotype).

Briefly SPR experiments were performed at 25° C. on a Biacore T200instrument employing a carboxymethyl dextran-coated (CM-5) chip. A mousemonoclonal anti-penta-histidine antibody (GE Healthcare) was immobilizedon the surface of the CM-5 sensor chip using standard amine-couplingchemistry. 140RU-376RU of His-tagged human or monkey FcγR proteins werecaptured on the anti-penta-histidine amine-coupled CM-5 chip (or in thecase of FcRn, about 155-299 RU of FcRn mutant contructs were immobilizedon a high density anti-myc coated Biacore chip) and stock solutions ofantibodies were injected at 20 μl/min for 2.5 min over the capturedproteins and serially diluted. mAb binding response was monitored and,for low affinity receptors, steady-state binding equilibrium wascalculated. Kinetic association (k_(a)) and dissociation (k_(d)) rateconstants were determined by processing and fitting the data to a 1:1binding model using Scrubber 2.0 curve fitting software. Bindingdissociation equilibrium constants (K_(D)) and dissociative half-lives(t_(1/2)) were calculated from the kinetic rate constants as: K_(D)(M)=k_(d)/k_(a); and t_(1/2) (min)=(In2/(60*k_(d)). Some KDs werederived using the steady state equilibrium dissociation constant; NB =nobinding observed.

An anti-CD3 antibody designated 9F7 having a heavy chain constant regionincluding hinge segments designated SEQ ID NO:1, SEQ ID NO:2, SEQ ID:3or SEQ ID NO:4 at residues 226-236, and of human IgG4 isotype, wastested for binding affinity to human FcγRI, RIIA, RIIB and RIII.Controls included the same antibody with wildtype IgG1 or IgG4 isotypes,and the same antibody with IgG4 isotype with a different modification ofthe hinge region (i.e. the hinge modification has positions 226-236occupied by CPPCPAPPVA-, the same sequences as in human IgG2, thereforereferred to as chimericFc). In the IgG4 chimeric Fc format, theremaining segments of the constant region are human IgG4. In the IgG1chimeric Fc format, the CH1 and CH3 segments are human IgG1 and CH2 ishuman IgG4.

The data show binding is reduced to background levels in all of thehinge-modified Antibodies 1-4 to each of human FcγRI, IIA, IIB and III.By contrast, binding of the IgG1 and IgG4 chimericFc antibodies isreduced to background levels for FcγRI and FcγIII, but is stillsignificant to FcγRIIA and RIIB. All of the hinge-modified Antibodies1-4 maintain binding to FcRn, comparable to IgG4 and chimeric hinge IgG4formats. See FIGS. 6-10.

Example 3 Cytotoxicity Analysis

U937 cells are a monocyte cell line expressing FcγI and FcγRIIA. U937cells were used as a positive killer effector control in the followingcytotoxicity assay. As such, the ability of antibodies with chimeric CHregions to kill U937 cells via Fc/FcγR interactions was tested. Calceinkilling assays were carried out using the following protocol: Human andcynomolgus Peripheral Blood Mononuclear Cells (PBMCs) were isolated overFicoll-Paque (GE Healthcare Life Sciences) or via Lymphocyte-Mammaldensity cell separation media (Cedarlane Laboratories), respectively.The isolated PBMCs were activated over a course of several days withmedia containing recombinant human IL-2 (30 U/ml) and T-cell activationbeads (anti-CD3/CD28 for human PBMC, anti-CD2/CD3/CD28 for cynomolgusPBMC). Activated T-cells were isolated from the PBMCs by centrifugation,then resuspended in 1 ml media. The magnetized beads were removed fromthe T-cells. Target cells (U937) were labeled with calcein, then washedand followed by incubation (10,000 cells per well) with 15-fold serialdilutions of purified Ab/sup and immortal CD8+ human T-cells (100,000cells/well) for 3 hr at 37 C (effector:target ratio—10:1) Followingincubation plates were centrifuged and the supernatant transferred toblack clear bottom plates for fluorescence analysis. Each EC50, definedas the molar concentration of antibody that induces 50% cytotoxicity,was determined using Prism (GraphPad Software, San Diego, Calif.).Values were calculated using a 4-parameter non-linear regressionanalysis (FIG. 11A).

The above experiments were performed with crude extracts of theantibodies in cell culture supernatants (FIG. 11A). Analogousexperiments were done with the same antibodies purified over standardaffinity columns or using the method described in Davis et al. (seeUS2010/0331527) for bispecific antibodies (FIG. 11B).

The data show that all of the hinge-modified antibodies 1-4 had onlybackground levels of cytotoxicity as was also the case for the IgG4chimericFc antibody. IgG1 and IgG4 wildtype antibodies showed strongcytotoxicity due to their ability to interact with FcγRI and FcγRIIA.See FIG. 11A and FIG. 11B.

Example 4 Activation of Jurkat Cells

This example tests whether an antibody can activate Jurkat cells (T cellleukemia cell line) transformed with an NFAT-luciferase construct thatacts as marker of activation. Activation requires an antibody to anchoron a HEK293 cells expressing FcγRIIA or FcγRIIB.

Jurkat/NFAT-Luc cells (50,000/well) were incubated with target cells(50,000/well) in the presence of serial dilutions of differentantibodies with (FIG. 12B, 12D) or without (FIG. 12A, 12C) antibody toan irrelevant antigen (FeID1)(1.5 mg/ml) for 4 h at 37 C. One-Glo(Promega) was added to measure luciferase activity.

The data show that all of the hinge-modified Antibodies 1-4 had onlybackground levels of activation. See. FIGS. 12A-12D. Accordingly, allhinge-modified Antibodies 1-4 lack ability to bind to FcγRIIA or IIB onHEC cells.

A positive control antibody of wildtype human IgG4 antibody showedstrong activation that was reduced to near background levels by theanti-FeID1 antibody, which competes for an anchoring site on HEK293.

To show that the lack of activation of Jurkat cells resulted fromdampening the FcγR binding ability of hinge-modified antibodies ratherthan an impaired ability of the antibodies to bind their CD3 target, anassay was performed with the antibodies cross-linked to a plate surface.Maxisorp plates were coated with a 2-fold serial dilution of differentAbs starting at 10 nM overnight at 4° C. Next day 50,000 Jurkat NFAT Luccells were added per well and media to make up the total volume to 100ul/well and incubate at 37° C. for 5 hours. 100 ul One-Glo (Promega) wasadded to measure luciferase activity. Transfer to opaque white nuncplates before reading luciferase activity. In this experiment all of thehinge-modified antibodies 1-4 showed similar activation to IgG4 isotypematched controls. See FIG. 13.

Example 5 ADCC Assay

In this assay, the hinge-modified antibodies have variable regions(3B9_VH) that bind the cell surface target antigen CD20 (Antibodies 5through 8, described above). CD20 positive target cells (Daudi) werelabeled with calcein, then washed and followed by incubation (10,000cells per well) with 6-fold serial dilutions of purified antibody andNK92_CD16V cells (NK92 cells engineered to express the higher affinity Vallele of FcγRIIIa at 50,000 cells/well) for 4 hr at 37 C(effector:target ratio—5:1). Target cell lysis was determined bymeasuring the calcein fluorescence in the supernatant. Percentcytotoxicity and EC50 were calculated analogously to that described inExample 3. FIG. 14 shows the hinge-modified antibodies do not mediateADCC activity (FIG. 14) against Daudi cells.

Example 6 PK Assessment of Anti-TM Monoclonal Antibody with ModifiedHinge

Antibodies having variable domains that bind to a multipasstransmembrane (TM) protein widely expressed in normal tissues andupregulated in various cancers were produced using well known techniques(see U.S. Pat. No. 5,057,604; WO 2011/143624; and WO 97/27873).

Assessment of the pharmacokinetic (PK) clearance rate: anti-TM antibodyhaving a modified hinge (Antibody 9) and a chimeric IgG4 Fc (Antibody10), as well as an isotype control with chimeric IgG4 Fc (Antibody 11)were assessed in C57BL/6 Wild-Type (WT) mice. For each anti-TM mAb orisotype control, cohorts of three mice were given a subcutaneous (s.c.)dose at 1 mg/kg. Mice were bled prior to the dosing and the serumsamples were designated as a pre-bleed or zero time point. All mice werebled at 6 hours, 1, 3, 7, 10 and 14 days post injection for PK analysis.Serum fractions from the bleeds were separated and frozen at −80° C.until analysis was conducted.

Determination of Total Drug Level in Sera by ELISA: Circulating anti-TMantibody levels were determined by total human antibody analysis usingan ELISA immunoassay. Briefly, a goat anti-human IgG polyclonal antibody(Jackson ImmunoResearch, #109-005-098) at 1 μg/ml in PBS was immobilizedon 96-well plates overnight and the plates were blocked with 5% BSA. Thedrug containing serum samples in six-dose serial dilutions and thereference standards of the respective antibodies in 12-dose serialdilutions were transferred to the prepared plates and incubated for onehour. The plate-bound anti-TM antibodies were then detected using a goatanti-human IgG polyclonal antibody conjugated with horseradishperoxidase (Jackson ImmunoResearch, #109-035-098). The plates weredeveloped with TMB substrate (BD Pharmingen, #51-2606KC, #15-2607KC)according to manufacturer's recommend protocol and signals of opticaldensity (OD) at 450 nm were recorded using a Perkin Elmer Victor X4Multimode Plate Reader. The anti-TM antibody concentrations in the serawere calculated based on the reference standard calibration curvegenerated using GraphPad Prism software.

C57BL/6 WT mice were given a single s.c. dose of 1 mg/kg of Antibody 9,Antibody 10 or Isotype control (Ab 11). Concentrations of total antibodywere determined at 6 time points over a 14-day time period. The totalanti-TM antibody concentrations for each antibody are summarized inTable 1.

TABLE 1 Serum Antibody Concentrations (Days 0, 0.25, 1, 3, 7, and 14)Total mAb concentration in mouse serum Antibody Time (d) Mean (μg/mL)+/−SD Antibody 9 0 ND ND Antibody 9 0.25 10 0.8 Antibody 9 1 13.3 0.8Antibody 9 3 10.6 0.7 Antibody 9 7 8.6 1   Antibody 9 14 5.3 0.4Antibody 9 0 ND ND Antibody 10 0.25 11.2 1.5 Antibody 10 1 14.4 0.9Antibody 10 3 11.0 1.6 Antibody 10 7 9.8 0.9 Antibody 10 14 6.4 0.5Antibody 11 0 ND ND Antibody 11 0.25 10.1 0.9 Antibody 11 1 12.0 0.4Antibody 11 3 10 0.6 Antibody 11 7 9 0.8 Antibody 11 14 5.1 0.2 Time =Time in days post single-dose injection; Day = Day of study; SD =Standard deviation; ND = Not detected

The mean concentration versus time profiles show that all threeantibodies achieved a maximum serum concentration (C_(max)) on day 1.Antibody 9, Antibody 10 and Isotype control had comparable C_(max)values of 13, 14 and 12 μg/mL, respectively. All three antibodiesexhibited a linear elimination with overlapping PK profiles. These PKprofiles are similar to those seen in previous studies for IgG1 andIgG4(5228P) isotype controls (data not shown). At day 14, Antibody 9 andisotype control has average drug levels around 5 μg/mL while Antibody 10had average drug levels around 6 μg/mL.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

SEQUENCE LISTING SEQ ID NO: 1 GGG-(233-236) PPCPAPGGG-GPSVFSEQ ID NO: 2 GG--(233-236) CPPCPAPGG--GPSVF SEQ ID NO: 3 G---(233-236)CPPCPAPG---GPSVF SEQ ID NO: 4 No_G-(233-236) CPPCPAP----GPSVFSEQ ID NO: 5 IgG4_GGG-(233-236)ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVTVPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPPCP APGGGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTYRVVSVLTVLH QDWLNGKEYK CKVSNKGLPSSIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDSDGSFFLYSRL TVDKSRWQEGNVFSCSVMHE ALHNHYTQKS LSLSLGKSEQ ID NO: 6 IgG4_GG--(233-236)ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVTVPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPPCP APGGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTYRVVSVLTVLH QDWLNGKEYK CKVSNKGLPSSIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDSDGSFFLYSRL TVDKSRWQEGNVFSCSVMHE ALHNHYTQKS LSLSLGKSEQ ID NO: 7 IgG4_G---(233-236)ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVTVPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPPCP APGGPSVFLFPPKPKDT LMISRTPEVT CVVVDVSQEDPEVQFNWYVD GVEVHNAKTK PREEQFNSTYRVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAKGQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKS LSLSLGK SEQ ID NO: 8 IgG4_No_G-(233-236)ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVTVPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPPCP APGPSVFLFPPKPKDT LMISRTPEVT CVVVDVSQEDPEVQFNWYVD GVEVHNAKTK PREEQFNSTYRVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAKGQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKS LSLSLGK SEQ ID NO: 9 IgG4*_GGG-(233-236)ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVTVPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPPCP APGGGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTYRVVSVLTVLH QDWLNGKEYK CKVSNKGLPSSIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDSDGSFFLYSRL TVDKSRWQEGNVFSCSVMHE ALHNRFTQKS LSLSLGKSEQ ID NO: 10 IgG4*_GG--(233-236)ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVTVPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPPCP APGGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTYRVVSVLTVLH QDWLNGKEYK CKVSNKGLPSSIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDSDGSFFLYSRL TVDKSRWQEGNVFSCSVMHE ALHNRFTQKS LSLSLGKSEQ ID NO: 11 IgG4*_G---(233-236)ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVTVPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPPCP APGGPSVFLFPPKPKDT LMISRTPEVT CVVVDVSQEDPEVQFNWYVD GVEVHNAKTK PREEQFNSTYRVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAKGQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNRFTQKS LSLSLGKSEQ ID NO: 12 IgG4*_No_G-(233-236)ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVTVPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPPCP APGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENNYKTTPPVLDS DGSFFLYSRL TVDKSRWQEGNVFSCSVMHE ALHNRFTQKS LSLSLGKSEQ ID NOS. 13-15 are wildtype human IgG1, IgG2 and IgG4 as shown in FIGS. 2-4.SEQ ID NO: 16 IgG1 GGGASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEPKSCDKTHTCPPCP APGGGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVSHEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGKEYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTCLVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRWQQGNVFSCSV MHEALHNHYT QKSLSLSPGK SEQ ID NO: 17 IgG1 GGASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEPKSCDKTHTCPPCP APGGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVSHEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGKEYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTCLVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRWQQGNVFSCSV MHEALHNHYT QKSLSLSPGK SEQ ID NO: 18 IgG1 GASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEPKSCDKTHTCPPCP APGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVSHEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGKEYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTCLVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRWQQGNVFSCSV MHEALHNHYT QKSLSLSPGK SEQ ID NO: 19 IgG1 no GASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEPKSCDKTHTCPPCP APG PSVFLFPPKP KDTLMISRTP EVTCVVVDVSHEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGKEYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTCLVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRWQQGNVFSCSV MHEALHNHYT QKSLSLSPGK SEQ ID NO: 20 IgG1* GGGASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEPKSCDKTHTCPPCP APGGGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVSHEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGKEYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTCLVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRWQQGNVFSCSV MHEALHNRFT QKSLSLSPGK SEQ ID NO: 21 IgG1* GGASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEPKSCDKTHTCPPCP APGGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVSHEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGKEYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTCLVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRWQQGNVFSCSV MHEALHNRFT QKSLSLSPGK SEQ ID NO: 22 IgG1* GASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEPKSCDKTHTCPPCP APGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVSHEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGKEYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTCLVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRWQQGNVFSCSV MHEALHNRFT QKSLSLSPGK SEQ ID NO: 23 IgG1* no GASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEPKSCDKTHTCPPCP APG PSVFLFPPKP KDTLMISRTP EVTCVVVDVSHEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGKEYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTCLVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRWQQGNVFSCSV MHEALHNRFT QKSLSLSPGK SEQ ID NO: 24: IgG2 GGGASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVT VPSSNFGTQT YTCNVDHKPS NTKVDKTVERKCCVECPPCP APGGGGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDPEVQFNWYVDG VEVHNAKTKP REEQFNSTFR VVSVLTVVHQ DWLNGKEYKCKVSNKGLPAP IEKTISKTKG QPREPQVYTL PPSREEMTKN QVSLTCLVKGFYPSDISVEW ESNGQPENNY KTTPPMLDSD GSFFLYSKLT VDKSRWQQGNVFSCSVMHEA LHNHYTQKSL SLSPGK SEQ ID NO: 25: IgG2 GGASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVT VPSSNFGTQT YTCNVDHKPS NTKVDKTVERKCCVECPPCP APGGGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDPEVQFNWYVDG VEVHNAKTKP REEQFNSTFR VVSVLTVVHQ DWLNGKEYKCKVSNKGLPAP IEKTISKTKG QPREPQVYTL PPSREEMTKN QVSLTCLVKGFYPSDISVEW ESNGQPENNY KTTPPMLDSD GSFFLYSKLT VDKSRWQQGNVFSCSVMHEA LHNHYTQKSL SLSPGK SEQ ID NO: 26 IgG2 GASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVT VPSSNFGTQT YTCNVDHKPS NTKVDKTVERKCCVECPPCP APGGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDPEVQFNWYVDG VEVHNAKTKP REEQFNSTFR VVSVLTVVHQ DWLNGKEYKCKVSNKGLPAP IEKTISKTKG QPREPQVYTL PPSREEMTKN QVSLTCLVKGFYPSDISVEW ESNGQPENNY KTTPPMLDSD GSFFLYSKLT VDKSRWQQGNVFSCSVMHEA LHNHYTQKSL SLSPGK SEQ ID NO: 27 IgG2 No GASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVT VPSSNFGTQT YTCNVDHKPS NTKVDKTVERKCCVECPPCP APGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDPEVQFNWYVDG VEVHNAKTKP REEQFNSTFR VVSVLTVVHQ DWLNGKEYKCKVSNKGLPAPIEKTISKTKG QPREPQVYTL PPSREEMTKN QVSLTCLVKGFYPSDISVEW ESNGQPENNY KTTPPMLDSD GSFFLYSKLT VDKSRWQQGNVFSCSVMHEA LHNHYTQKSL SLSPGK SEQ ID NO: 28: IgG2* GGGASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVT VPSSNFGTQT YTCNVDHKPS NTKVDKTVERKCCVECPPCP APGGGGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDPEVQFNWYVDG VEVHNAKTKP REEQFNSTFR VVSVLTVVHQ DWLNGKEYKCKVSNKGLPAPIEKTISKTKG QPREPQVYTL PPSREEMTKN QVSLTCLVKGFYPSDISVEW ESNGQPENNY KTTPPMLDSD GSFFLYSKLT VDKSRWQQGNVFSCSVMHEA LHNRFTQKSL SLSPGK SEQ ID NO: 29: IgG2* GGASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVT VPSSNFGTQT YTCNVDHKPS NTKVDKTVERKCCVECPPCP APGGGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDPEVQFNWYVDG VEVHNAKTKP REEQFNSTFR VVSVLTVVHQ DWLNGKEYKCKVSNKGLPAPIEKTISKTKG QPREPQVYTL PPSREEMTKN QVSLTCLVKGFYPSDISVEW ESNGQPENNY KTTPPMLDSD GSFFLYSKLT VDKSRWQQGNVFSCSVMHEA LHNRFTQKSL SLSPGK SEQ ID NO: 30 IgG2* GASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVT VPSSNFGTQT YTCNVDHKPS NTKVDKTVERKCCVECPPCP APGGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDPEVQFNWYVDG VEVHNAKTKP REEQFNSTFR VVSVLTVVHQ DWLNGKEYKCKVSNKGLPAP IEKTISKTKG QPREPQVYTL PPSREEMTKN QVSLTCLVKGFYPSDISVEW ESNGQPENNY KTTPPMLDSD GSFFLYSKLT VDKSRWQQGNVFSCSVMHEA LHNRFTQKSL SLSPGK SEQ ID NO: 31 IgG2* No GASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVT VPSSNFGTQT YTCNVDHKPS NTKVDKTVERKCCVECPPCP APGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDPEVQFNWYVDG VEVHNAKTKP REEQFNSTFR VVSVLTVVHQ DWLNGKEYKCKVSNKGLPAP IEKTISKTKG QPREPQVYTL PPSREEMTKN QVSLTCLVKGFYPSDISVEW ESNGQPENNY KTTPPMLDSD GSFFLYSKLT VDKSRWQQGNVFSCSVMHEA LHNRFTQKSL SLSPGK

What is claimed is:
 1. An immunoglobulin heavy chain comprising aconstant region, wherein positions 233-236 within a hinge domain are G,G, G and unoccupied; G, G, unoccupied, and unoccupied; G, unoccupied,unoccupied, and unoccupied; or all unoccupied, with positions numberedby EU numbering.
 2. The immunoglobulin heavy chain of claim 1 that ishuman IgG4 isotype.
 3. The immunoglobulin heavy chain of any precedingclaim, wherein positions 226-229 are CPPC.
 4. The immunoglobulin heavychain of any preceding claim, wherein the hinge domain amino acidsequence comprises CPPCPAPGGG-GPSVF (SEQ ID NO:1), CPPCPAPGG--GPSVF (SEQID NO:2), CPPCPAPG---GPSVF (SEQ ID NO:3), or CPPCPAP----GPSVF (SEQ IDNO:4).
 5. The immunoglobulin heavy chain of any preceding claim, whereinthe constant region has an amino acid sequences comprising SEQ ID NO:5,6, 7 or 8 or a variant thereof having up to five insertions deletions,substitutions or insertions.
 6. The immunoglobulin heavy chain of anypreceding claim wherein the constant regions comprises SEQ ID NO: 5, 6,7 or
 8. 7. The immunoglobulin heavy chain of any preceding chain whereinthe constant region consists of SEQ ID NO: 5, 6, 7 or
 8. 8. Theimmunoglobulin heavy chain of claim 1 comprising from N-terminal toC-terminal the hinge domain, a CH2 domain and a CH3 domain.
 9. Theimmunoglobulin heavy chain of claim 1, comprising from N-terminal toC-terminal a CH1 domain, the hinge domain, a CH2 domain and a CH3 domain10. The immunoglobulin heavy chain of claim 8 or 9, wherein the CH1region, if present, remainder of the hinge region, if any, CH2 regionand CH3 region are the same human isotype.
 11. The immunoglobulin heavychain of claim 8 or 9, wherein the CH1 region, if present, remainder ofthe hinge region, if any, CH2 region and CH3 region are human IgG1. 12.The immunoglobulin heavy chain of claim 8 or 9, wherein the CH1 region,if present, remainder of the hinge region, if any, CH2 region and CH3region are human IgG2.
 13. The immunoglobulin heavy chain of claim 8 or9, wherein the CH1 region if present, remainder of the hinge region, ifany, CH2 region and CH3 region are human IgG4.
 14. The immunoglobulin ofany preceding claim, wherein the constant region has a CH3 domainmodified to reduce binding to protein A.
 15. The immunoglobulin heavychain of any preceding claim linked at the N-terminus to a heavy chainvariable region.
 16. The immunoglobulin heavy chain of claim 11 duplexedwith an immunoglobulin light chain.
 17. The immunoglobulin heavy chainof claim 11 duplexed with an immunoglobulin light chain as a heterodimercomprising two immunoglobulin heavy chains and two light chains.
 18. Theimmunoglobulin of claim 13, wherein the two heavy chains are the same.19. The immunoglobulin of claim 13, wherein the two heavy chains aredifferent.
 20. The immunoglobulin heavy chain of any preceding claimlinked at the N-terminus to a binding polypeptide.
 21. Theimmunoglobulin heavy chain of claim 20 linked via a linker to thebinding polypeptide.
 22. The immunoglobulin heavy chain of claim 20 or21, wherein the binding polypeptide is an extracellular domain.